Optogenetics holds tremendous potential for restoring vision to individuals with late-stage retinal degeneration, particularly those patients who have lost most of their photoreceptors. One promising therapeutic strategy is to express a light-sensitive protein in non-photosensitive bipolar cells by gene therapy. Current approaches are limited by inefficient bipolar targeting and expression, and require application of potentially phototoxic levels of blue-green light to stimulate the optogenetic actuator. The objective of the present proposal is to overcome these challenges by utilizing directed evolution and synthetic biology to engineer an AAV-based delivery system to target red-shifted optogenetic devices to both ON and OFF bipolar cells, and to employ this system to treat blindness in mice. In Aim 1, we will use directed evolution to engineer new AAV serotypes capable of highly efficient bipolar AAV infection after injection into the vitreous humor. In Aim 2, we will utilize a novel technolog called CRE-seq to engineer thousands of compact, ON bipolar-specific promoters that exhibit excellent specificity and a wide range of expression strengths. In addition, we will engineer an AAV-deliverable synthetic gene circuit to target an optogenetic inhibitor specifically to OFF bipolar cells. In Aim 3, we will combine the tools developed in Aims 1 and 2 with the use of red-shifted optogenetic devices to restore functional vision to rd1 mutant mice. We recently discovered the enzyme responsible for the 'rhodopsin- porphyropsin' switch in vertebrates, and we will use this enzyme to red-shift optogenetic devices, making them sensitive to far red light (> 650 nm). This therapeutic approach has the potential to dramatically improve light- sensitivity in the rescued mice and will avoid the retinal damage associated with high-intensity blue light exposure, thereby permitting unprecedented levels of functional restoration and setting the stage for future trials in human patients.